Germline mutations provide the substrate for evolution, whether mediated by natural selection or random genetic drift. The inheritance of advantageous mutations increases the chance of survival and reproduction while the inheritance of deleterious mutations causes or predisposes to illness. Germline mutations in humans can neither be observed directly nor manipulated experimentally. However, inferences about the germline mutational process are possible by detailed analysis with attention to defining and correcting for biases in the data. The human factor IX gene is an advantageous system for examining the mutational process in the human germline. The goal of this grant application is to extend the analysis of the factor IX gene in order to gain further novel insights into the mutational process during both the recent and the more distant past. Molecular analysis of the factor IX gene in patients with hemophilia B will define germline mutations that generally have occurred within the past 150 years. In contrast to most genes, the observed pattern of mutation in patients with hemophilia B is similar to the underlying pattern of mutation. In addition, molecular analyses of: (i) intronic sequence changes in humans, (ii) intronic sequence changes in primates, and (iii) exonic sequence changes in nonmammals will define essentially neutral mutations that generally have occurred thousands, millions, and hundreds of millions of years ago, respectively. In the present application, the following questions will be addressed: (i) How does the sex ratio of recent germline mutation depend on the type of mutation and the age of the parent?; (ii) How frequent is somatic mosaicism and how likely is it to occur at a frequency of 10-1, 10-2, and 10-3?; (iii) Does the pattern of recent human factor IX germline mutation vary with ethnicity or geography?; (iv) What is the nature of the mutations that affect both factor IX mRNA structure and occur outside the regions of likely functional significance?; (v) Do germline mutations tend to arise in clusters?; (vi) Is the pattern of intronic polymorphism in Caucasians, Asians, Blacks, and Amerindians the same as the inferred underlying pattern of germline mutation during the past 150 years?; (vii) Is the average pattern of fixed germline mutations in the factor IX gene in eight great apes and Old World monkeys the same in each lineage?; and (viii) Are the amino acids conserved in factor IX during nonmammalian evolution the same amino acids that are mutated in humans with hemophilia B?